4.5.4.1. Preliminary tests

To facilitate and optimize efficacy test, it is recommended to perform dose finding and tolerance test on caged bees under controlled conditions in the laboratory. See section 3.6.3. ‘Bioassays to quantify the susceptibility of the varroa mite to acaricides’ and the BEEBOOK paper on toxicology methods (Medrzykci et al., 2013). The highest concentration/quantity tolerated by the honey bees can be used as an indication for concentrations or quantities that can be used in subsequent dose-titration as well as dose-confirmation or field studies. Dose-titration studies should aim at identifying the minimum effective and maximum tolerated levels of active substance reaching bees and parasites and thus help establishing the dosage and dosing interval of the product. Implementation of dose-finding studies, carried out under controlled laboratory conditions is preferred, e.g. using 10 workers per cage, 3 cages per concentration, 3 untreated controls and one replicate, i.e. the studies should be carried out twice. See the BEEBOOK paper on toxicological methods (Medrzycki et al., 2013).

Small scale outdoor pilot studies to confirm dose, efficacy and tolerance should be considered before large scale field studies are performed. It is thus possible to validate the results obtained in the laboratory in a situation closer to that of the field, but with a high reproducibility since variables can be better controlled in these small units compared to full size colonies. It also allows for troubleshooting before the investment in the full scale test is done. A minimum of five untreated control and five treated test colonies should be used. To ensure reproducibility in the pilot studies, colonies should be comparable with respect to environment, type and size of hive, level of varroa infestation, treatment history, age of queen, relatedness of queens (sister queens can be used to decrease variability between replicates, in contrast, unrelated queens can be used to consider a wider range of genotypes), presence of brood, and age distribution of worker bees.